A large number of dental implant rehabilitation procedures are performed every year. In contrast to the vast majority of cases where implant treatment is successful, a certain number of patients develop peri-implant disease (PI). In some cases, non-surgical treatment with mechanical debridement and flushing with 3% hydrogen peroxide may be a sufficiently effective treatment. In cases of persisting peri-implant disease, resective surgery in combination with surface debridement is often performed. By surgical correction of osseous defects (e.g. bone peaks) at the diseased implant site, pocket depths can be reduced and provide for a soft tissue morphology that facilitates oral hygiene. However, certain patients do not respond sufficiently well to treatment, and in spite of good plaque control and minimal inflammation of the peri-implant mucosa, symptoms including suppuration and progressive bone loss may recur in some cases. The reasons for such relapses are not known. A desire for improved understanding of the etiology of peri-implant disease and for the development of more sensitive diagnostic tools allowing for earlier detection and interventions is at hand; thus, increasing the predictability of implant treatment in susceptible patients. Moreover, in order to increase the survival rate of implants presenting signs of bone loss, clinical intervention at an early stage of disease progression is desirable. This requires early establishment of possible ongoing bone resorption, and therefore, more sensitive techniques are required.
Bone resorption is mediated by bone resorption cells, osteoclasts, which are formed by mononuclear phagocytic cells. New bone replacing the lost bone is deposited by bone-forming cells, osteoblasts, which are formed by mesenchymal stromal cells. Various other cell types that participate in the remodeling process are tightly controlled by systemic factors (e.g., hormones, lymphokines, growth factors and vitamins) and local factors (e.g., cytokines, adhesion molecules, lymphokines and growth factors) (WO2012/061907).
The diagnosis of peri-implant disease is generally based on clinical measurements combined with radiographic evidence of bone loss. Peri-implantitis is often clinically translated into formation and deepening of pockets, breakdown of the peri-implant epithelial seal, bleeding on probing (BoP), purulence and progressive bone loss. These diagnostic methods are often used in combination for diagnosis of peri-implant disease as indicators of extensive pathologic changes in the implant-supporting tissue. The limited sensitivity and/or specificity of such diagnostic methods make early detection of pathologic changes difficult.
The viability of using analysis of genetic markers in the gingival crevicular fluid in plaque samples as a potential prognostic and diagnostic tool for peri-implant disease has been studied by a number of authors with variable results.
An often studied marker is Interleukin-1β (IL-1β), which is a pro-inflammatory cytokine involved in several biologic processes, including immune regulation, inflammation and connective tissue metabolism. IL-1β stimulates bone resorption and inhibits bone formation (Panagakos et al., Int J Oral Maxillofac Implants 1996, 11:794-799). IL-1β is produced mainly by macrophages but also by other cells including neutrophilic granulocytes. Several studies have shown the presence of IL-1β in the crevicular fluid around implants presenting signs of peri-implant disease. Significantly elevated levels have been reported for peri-implantitis compared to healthy sites (Panagakos et al., Int J Oral Maxillofac Implants 1996, 11:794-799; Kao et al., Int J Oral Maxillofac Implants 1995, 10:696-701; Murata et al., Clin Oral Impl Res 2002, 13:637-643) and compared to peri-implant mucositis sites (Murata et al., Clin Oral Impl Res 2002, 13:637-643), and also when comparing subjects with early and advanced signs of peri-implantitis. However, Hultin et al. (Clin Oral Impl Res 2002, 13:349-358) showed contradictory results with no difference in IL-1β expression between peri-implantitis and healthy sites.
Interleukin-8 (IL-8) is a proinflammatory marker and chemotactic factor for neutrophils. It participates in the regulation of the innate immune response to microbial invasion in periodontitis (Nassar et al., Infection and Immunity 2002, 268-276; Goutoudi et al., Int J Dent 2012; 2012:362905) and peri-implantitis (Petkovic et al., Int J Oral Maxillofac Surg 2010, 39(5):478-85). Nowzari et al. (Clin Implant Dent Relat Res 2008, 10(3):166-173) studied cytokine presence around implants and teeth in healthy subjects, and found a two-fold increase of IL-8 around implants compared with teeth.
Interleukin-6 (IL-6) is a multifunctional cytokine produced by various cells to regulate hematopoiesis, inflammation, immune responses, and bone homeostasis (Yoshitake et al., J Biol Chem 2008, 283:11535-11540). The level of IL-6 in saliva samples from subjects with peri-implant disease was significantly elevated compared with saliva samples from healthy subjects in a study by Liskmann et al. (Int J Oral Maxillofac Implants 2006, 21(4):543-50). Konttinen et al (Int J Periodontics restorative Dent 2006, 26:135-141) measured statistically higher levels of IL-6 at failing implants with peri-implantitis compared with healthy implant sites.
Osteoclasts are bone resorbing cells originating from progenitor cells of the monocyte/macrophage lineage. The process of osteoclastogenesis is coordinated by receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG), which are members of the tumor necrosis factor super family. While RANKL induces osteoclastogenesis, its antagonist OPG inhibits the formation of osteoclasts (Boyle et al., Nature 2003, 423:337-342; Leibbrandt et al., Ann NY Acad Sci 2008, 1143:123-150). OPG has also been found to decrease osteoclast apoptosis (Chamoux et al., J Cell Physiol. 2008, 216(2):536-42). RANKL and OPG are secreted by osteoblasts, fibroblasts and endothelial cells (Corralini et al., J Cell Physiol. 2011, 226(9):2279-2286). RANKL is also expressed by activated T cells (Saidenberg-Kermanac'h et al., Eur Cytokine Netw. 2002, 13(2):144-53). It has been suggested that an imbalance in the equilibrium between OPG and RANKL may be related to diseases involving bone destruction such as periodontitis. For example, Bostanci et al., (J Clin Periodontol 2007, 34:370-376) showed that RANKL and OPG levels in the gingival crevicular fluid were oppositely regulated in periodontitis but not in gingivitis; hence, a significantly lower RANKUOPG ratio was recorded in gingival crevicular fluid around healthy teeth compared to teeth presenting various degrees of periodontal disease. Nevertheless, two studies of OPG and soluble RANKL in crevicular fluid around implants have failed to show statistically significant correlations with clinical parameters (Arikan et al., Clin Oral Impl Res 2008, 19:283-288; Monov et al., Clin Implant Dent Relat Res 2006, 8(3):135-141). However, in both studies numerous samples were outside the detection limit of the assay and either were excluded from the statistical calculations or accounted as 0. Hence, the authors conclude that the presented data should be interpreted with regard to these limitations. The role of OPG for the pathogenesis of arthritis has also been studied. For example, Liu et al. (Chin Med J (Engl). 2010, 123(11):1407-12) reported that the level of circulating OPG was elevated in subjects with early rheumatoid arthritis. It has been shown that Wnt proteins can promote maintenance and proliferation of stem cells (Willert et al., Nature 2003, 423(6938):448-52), and Wnt signaling plays a dominant role for osteoblastogenesis (for review, see Yavropoulou et al., Hormones, 2007, 6(4):279-294).
Cathepsin K (CatK) is a bone resorption marker, which is highly expressed in active osteoclasts. Higher CatK expression in peri-implantitis sites compared to healthy sites has been demonstrated (Strbac et al., J Clin Periodontol 2006; 33:302-308).
Osteocalcin (OC) is a calcium-binding protein of bone involved in bone mineralization and calcium homeostasis. Murata et al. (Clin Oral Impl Res 2002, 13:637-643) showed increases in OC expression in peri-implant crevicular fluid (PICF) from mucositis sites compared to healthy sites, while no differences in OC levels were seen between peri-implantitis sites and mucositis or healthy implant sites.
Matrix metalloproteinases (MMPs) are proteolytic enzymes involved in degradation and removal of collagen from damaged tissue. MMPs are secreted by cells residing in the inflammatory sites in response to stimuli such as lipopolysaccharide and cytokines (Aboyoussef et al., Int J Oral Maxillofac Implants 1998, 13:689-696). Collagenases and gelatinases are two sub-families of the MMP superfamily. Findings by Kivelä-Rajamäki et al. (Clin Oral Impl Res 2003, 14:158-165) indicated that increased levels of MMP-8 (collagenase-2) may be associated with the active phase of inflammatory peri-implant disease. The expression of MMP-9 (gelatinase B) has also been studied; while Ma et al. (Clin Oral Impl Res 2003, 14:709-713) showed an association between MMP-9 and bone levels, Aboyoussef et al. (Int J Oral Maxillofac Implants 1998, 13:689-696) failed to show any significant differences between healthy and peri-implantitis sites.
The imbalance between MMPs and tissue inhibitors of matrix metalloproteinases (TIMPs) is considered to trigger the degradation of extracellular matrix, basement membrane, and alveolar bone, and thus to initiate periodontal disease (Sorsa et al., Oral Diseases 2004, 10: 311-318). It has been suggested that salivary MMP-8, TIMP-1 and especially their ratios are potential candidates in the detection of advanced periodontitis (Gursoy et al., Clin Periodontol 2010, 37:487-493).
The plasminogen system is of central importance in extracellular proteolysis in physiological as well as pathological tissue remodeling (reviewed by Collen, Thromb Haemost 1999, 82:259-270). Plasmin is a broadly active protease that is capable of degrading many extracellular proteins as well as activating latent collagenase and other metalloproteinase (Werb et al., New Eng J Med 1977, 296:1017-1023; Matrisian, Bioessays 1992, 14:455-463). Plasmin acts directly on the extracellular matrix (ECM) by cleaving non-collagenous ECM proteins and also indirectly by activating proforms of a whole range of other enzymes, among them the matrix metalloproteinases (MMPs), with specificity for different connective tissue proteins. Through the interaction between the plasminogen system and other tissue degrading systems, plasminogen represents an important dormant proteolytic potential, and strict control of its activation is important for maintaining the integrity of the tissues. Plasmin is formed from its inactive precursor plasminogen by plasminogen activators (serine proteases of which two types have been identified: urokinase type, u-PA, and tissue type, t-PA), which are specifically inhibited by the plasminogen activator inhibitors (PAI-1 and PAI-2), through the formation of bimolecular 1:1 covalent complexes. The levels of tPA as well as PAI-2 have been shown to be higher in gingival crevicular fluid (GCF) from inflamed than healthy sites (Kinnby, Biol Chem 2002, 383:85-92). A relatively increased level of PAI-2 has been associated with tissue-protective functions in pregnancy as well as periodontitis (Kinnby et al., J Periodont Res 1996, 31:271-277; Olofsson et al., J Periodont Res 2002, 37:60-65).
Different treatment alternatives for peri-implant disease have been proposed. It has been suggested that non-surgical therapy (e.g. surface debridement without access surgery) may be successful in cases of peri-implant mucositis, but appears to be less effective for sites presenting peri-implantitis (Renvert et aL, J Clin Periodontol 2008, 35 (Suppl 8):305-315). Clinical data suggests that surgical treatment—e.g. open debridement including surface decontamination in combination with systemic antibiotics—may be a viable treatment option for peri-implantitis lesions (Claffey et al., J Clin Periodontol 2008, 35 (Suppl 8): 316-332). However, to date no common therapy exists, and advanced peri-implantitis remains difficult to treat. The marginal bone around the implant crestal region is usually a significant indicator of implant health. The level of the crestal bone may be measured from the crestal position of the implant at the initial implant surgery. The most common method to asses bone loss is by radiographic evaluation. The bone level can thus be measured on the radiographs and can be defined as the distance from the junction between the fixture and its abutment to the crest of the marginal bone mesially and distally to the implants (Ahlqvist et al., Int J Oral Maxillofac Implants 1990, 5(2):155-163). Of course, conventional radiographics only monitor the mesial or distal aspect of bone loss around the implant body (Misch et aL, Implant Dentistry 2008, 17(1):5-15). Lack of unambiguous information on ongoing bone loss may result in unnecessary or even incorrect treatment of peri-implant disease. The peri-implant bone level is determined from radiographs usually taken at the time of diagnosis The bone level is compared with what is considered normal, and one or more radiographs taken at earlier time points are used to assess the bone loss. However, radiographs provide a stationary image of the bone situation; hence, evidence of bone demineralization does not necessarily imply ongoing disease activity. This holds true also for periodontal bone levels, and data on progression of periodontitis do not demonstrate a continuous process but instead bursts of activity (exacerbation), remission and periods of inactivity (Hall et al., Eur J oral Implantol 2011, 4(4):371-382). In addition, the limited sensitivity of radiographs seldom allow for detection of the very early stages of the pathological bone degradation processes involved in several diseases. Moreover, it is important that all radiographic examinations be performed using appropriate and reproducible projection techniques. The precision in measurements performed on radiographs is low, especially when related to small average bone loss, and it indicates the difficulties involved in the interpretation of them. Furthermore, the bone loss rate can only be measured within a long period of time, typically one year (Ahlqvist et al., Int J Oral Maxillofac Implants 1990, 5(2):155-163), and involves exposing patients to frequent radiation. Therefore, it seems likely that establishment of ongoing bone degradation in peri-implantitis and periodontitis patients is a prerequisite for increased accuracy of individualized patient treatment.
Implant success allow for a rate of bone loss not exceeding 1.5 mm first year of implant loading and 0.2 mm/year thereafter. A higher progressive bone loss is indicative of peri-implantitis, implant overload or suboptimal implant placement hampering the healing capability at the implant site. Peri-implantitis and periodontitis progresses in a bi-phasic manner, where an active phase with bone loss is followed by a passive phase with no or insignificant bone loss and so on (Hall et al., Eur J oral Implantol 2011, 4(4):371-382). Therefore, if the bone loss rate should remain constant at a certain level, it will take a certain time before the implant or the tooth no longer is supported by anchoring bone, which depends on the length of the implant or tooth and the load bearing capability of the remaining surrounding bone.